1. Field of the Invention
The present invention relates to a measuring unit, to be employed in a measuring apparatus that causes a light beam to be totally internally reflected at an interface between a thin film layer, which is in contact with a measurement target such as a sample, and a dielectric block, to generate evanescent waves, and that measures changes in the intensity of the totally internally reflected light beam due to the evanescent waves, to analyze the sample. Particularly, the present invention relates to a one dimensional measuring unit, which is equipped with a plurality of measurement stations arranged in the longitudinal direction thereof.
2. Description of the Related Art
Surface plasmon sensors are known, as a type of measuring apparatus that utilizes evanescent waves. In metals, free electrons oscillate in groups to generate compression waves, called plasma waves. The compression waves which are generated at the surface of metals are called surface plasmon, when quantized. Various known surface plasmon sensors utilize a phenomenon, in which the surface plasmons are excited by light waves, to analyze properties of samples. Particularly well known surface plasmon sensors are those of a Kretschmann configuration (as disclosed in Japanese Unexamined Patent Publication No. 6(1994)-167443, for example).
Surface plasmon sensors of the Kretschmann configuration basically comprise: a dielectric block, shaped as a prism, for example; a metal film, formed on one surface of the dielectric block and which is brought into contact with a sample; a light source for emitting a light beam; an optical system for causing the light beam to enter the dielectric block at various angles of incidence so that total internal reflection conditions are satisfied at an interface of the dielectric block and the metal film; a photodetecting means for detecting the intensity of the light beam, which has been totally reflected at the interface; and a measuring means for measuring the state of surface plasmon resonance, based on detection results obtained by the photodetecting means.
In order to obtain various angles of incidence for the light beam, a comparatively thin incident light beam may be caused to impinge upon the interface while changing the angle of incidence. Alternatively, a comparatively thick incident light beam may be caused to impinge upon the interface in the form of convergent light or divergent light, so that the incident light beam includes components impinging upon the interface at various angles. In the former case, the light beam which is reflected from the interface at an angle, which varies as the angle of incidence changes, may be detected by a small photodetector which is moved in synchronization with the change of the angle of incidence, or by an area sensor that extends in the direction coincident with the angles of reflected light. In the latter case, an area sensor, which extends in directions such that all the components of light reflected from the interface at various angles can be detected thereby, may be employed.
In a surface plasmon sensor of the construction described above, when a light beam impinges upon the metal film at a particular angle of incidence θsp greater than or equal to the angle of total internal reflection, evanescent waves having an electric field distribution in a sample which is in contact with the metal film are generated, and surface plasmon is excited at an interface between the metal film and the sample. When the wave vector of the evanescent light is equal to the wave number of the surface plasmon and wave number matching is established, the evanescent waves and the surface plasmon resonate and light energy is transferred to the surface plasmon, whereby the intensity of light reflected in total internal reflection at the interface of the dielectric block and the metal film sharply drops. The sharp intensity drop is generally detected as a dark line by the photodetector.
When the wave number of the surface plasmon can be known from the angle of incidence θsp at which the phenomenon of attenuation in total internal reflection (ATR) takes place, the dielectric constant of the sample can be obtained. That is,
            K      sp        ⁡          (      ω      )        =            ω      c        ⁢                                                      ɛ              m                        ⁡                          (              ω              )                                ⁢                      ɛ            s                                                              ɛ              m                        ⁡                          (              ω              )                                +                      ɛ            s                              wherein Ksp represents the wave number of the surface plasmon, ω represents the angular frequency of the surface plasmon, c represents the speed of light in a vacuum, and ∈m and ∈s respectively represent the dielectric constants of the metal and the sample.
When the dielectric constant ∈s of the sample is known, the refractive index and the like of the sample can be determined on the basis of a predetermined calibration curve or the like. As a result, properties of the sample related to the refractive index, such as the dielectric constant, can be determined, by determining the angle θsp at which attenuated total reflection occurs (hereinafter, referred to as “attenuated total reflection angle θsp”).
As a similar type of sensor that utilizes evanescent waves, there is known a leaky mode sensor as described in, for instance, “Surface Refracto-Sensor using Evanescent Waves: Principles and Instrumentations” by Takayuki Okamoto, Spectrum Researches, Vol. 47, No. 1 (1998), pp. 19-28. The leaky mode sensor basically comprises: a dielectric block, shaped as a prism, for example; a cladding layer, formed on one surface of the dielectric block; an optical waveguide layer, which is formed on the cladding layer and which is brought into contact with a sample; a light source for emitting a light beam; an optical system for causing the light beam to enter the dielectric block at various angles of incidence so that total internal reflection conditions are satisfied at an interface of the dielectric block and the cladding layer; a photodetecting means for detecting the intensity of the light beam, which has been totally reflected at the interface; and a measuring means for measuring the state of excitation of a waveguide mode, based on detection results obtained by the photodetecting means.
In a leaky mode sensor of the construction described above, when the light beam is caused to impinge upon the cladding layer through the dielectric block at an angle greater than or equal to an angle of total internal reflection, evanescent waves are generated in the optical waveguide layer and an evanescent wave having a particular wave number comes to propagate through the optical waveguide layer in a waveguide mode. When the waveguide mode is thus excited, almost all the incident light which generates the evanescent wave having a particular wave number is taken into the optical waveguide layer and accordingly, the intensity of light reflected in total internal reflection at the interface of the dielectric block and the clad layer sharply drops. Because the wave number of light to be propagated through the optical waveguide layer depends upon the refractive index of the sample on the optical waveguide layer, the refractive index and properties of the sample related to the refractive index can be determined, based on the attenuated total reflection angle θsp.
The aforementioned surface plasmon sensors and leaky mode sensors may be utilized to perform random screening in the field of pharmaceutical manufacture. In random screening, specific substances that bond with a desired sensing substance are sought. In this case, the sensing substance is disposed on the thin film (the metal film in the case of a surface plasmon sensor, and the optical waveguide layer and the cladding layer in the case of a leaky mode sensor). Then, various solutions of test targets (sample liquids) are added to the sensing substance. Each time that a predetermined amount of time passes, the attenuated total internal reflection angle θsp is measured. If the test target binds with the sensing substance, the refractive index of the sensing substance changes over time due to the bond. Accordingly, whether the test target is bonding with the sensing substance, that is, whether the test target is the specific substance that bonds with the sensing substance, can be determined by measuring the attenuated total internal reflection angle θsp at predetermined time intervals, thereby measuring whether the attenuated total reflection angle θsp changes. A combination of an antigen and an antibody is an example of the combination of the specific substance and the sensing substance. Alternatively, a combination of an antibody and another antibody may be the combination of the specific substance and the sensing substance. Measurement regarding whether a rabbit antihuman IgG antibody, as a sensing substance, bonds with an antihuman IgG antibody, as a specific substance, and quantitative analysis of the bond, are specific examples of measurement.
Note that it is not necessary to detect the attenuated total reflection angle θsp itself, in order to measure bonding states between test targets and sensing substances. For example, a sample liquid may be added to a sensing substance, then the variation in the attenuated total reflection angle θsp may be measured. The bonding state may be measured, based on the degree of the variation of the attenuated total reflection angle θsp.
Meanwhile, there are known sensors that perform measurements by employing a planar measuring chip, on which a sensing substance is fixed. Liquid samples are continuously supplied to the measuring chip via a flow path mechanism (as disclosed in, for example, Japanese Unexamined Patent Publication No. 2000-065731). In this type of sensor, when measuring a bonding state between the sensing substance and a specific substance, fresh liquid samples are constantly supplied to the measuring chip. Therefore, the concentration of the test target within the sample liquid remains constant, and measurement of the bonding state can be performed favorably. In addition, if bonding continues to occur after measurement of the bonding state between the sensing substance and the specific substance, a liquid sample that does not include the specific sample may be caused to flow over the measuring chip, on which the bonded compound is fixed. Thereby, a dissociating state of the sensing substance and the specific substance can be measured. Further, in cases that a gas, or a liquid sample in which gas is included, is employed as a sample, the sample can be easily supplied to the measuring chip, by use of the flow path mechanism.
In recent years, various types of solvent mediums are being employed, accompanying the diversity of reactions to be detected. Among the solvent mediums, there are those, such as water, which are likely to evaporate. Evaporation of water, when used as a solvent medium, changes the refractive index of the measurement sample, which changes the measurement signal. Therefore, there are cases that accurate measurement becomes difficult. In these cases, it is possible to minimize evaporation of the measurement sample and thereby stabilize the measurement signal, by providing the aforementioned flow path mechanism.
Various advantageous are obtained by providing the flow path mechanism, as described above. However, there are drawbacks, such as a long piping system being required to supply the samples onto the measuring chip, and a large amount of the sample being necessary.
Therefore, a so-called one dimensional measurement unit has been proposed in U.S. Patent Application Publication No. 20040205058. This measuring unit comprises: an elongate dielectric block, which is transparent with respect to a light beam; a thin film layer, formed on a flat surface of the dielectric block; and a flow path forming member, which is in close contact with the thin film layer of the dielectric block, for forming a plurality of flow paths in the longitudinal direction of the dielectric block on the thin film layer, with intervals therebetween. The flow paths are designated as measurement paths on the thin film layer. The ends of each flow path are in communication with an entrance and an exit of the flow path forming member, to form supply paths and discharge paths. Pipettes are provided at the entrance and the exit of the flow path forming member, to supply and discharge liquid samples.
Liquid samples are enabled to be supplied to the entrance of the flow path forming member of the one dimensional measuring unit as described above by an external liquid supplying component, such as a pipette chip. Therefore, a flow path mechanism for supplying a sample to the thin film layer is provided, while the necessity of the long piping system is obviated. Accordingly, it is possible to perform measurements with a small amount of the sample.
However, in order to accurately set the long measurement unit at a predetermined position, protrusive portions that protrude from the two ends of the measuring unit are held to handle the measuring unit. Therefore, a holding mechanism must be capable of accurate movement in the X, Y, and Z directions, which complicates the holding mechanism. In addition, positional displacement during readout becomes likely to occur, which is not favorable from a practical point of view.